Project Summary Complex host-microbe interactions that define immunological health and disease may begin in utero and therefore, chronic inflammatory diseases could be prevented before birth. Our bodies are inhabited by vast microbial ecosystems, which are shaped by time, diet, and environmental exposures. The microbiome also shapes the host: it regulates immune development, homeostasis, and susceptibility to disease development at mucosal sites and organ systems. The long-held assumption that the uterine environment is sterile has been challenged by many groups, because bacteria were detected in the placenta, fetal membranes, amniotic fluid, and human meconium (the first neonatal bowel movement composed of swallowed amniotic fluid). These studies are fundamental, because ecologically we know that pioneering species colonizing a pristine habitat can play a substantial role in defining ecosystem conditions and the trajectory of primary succession, or the process of species accumulation and replacement over time. Human immune development also occurs in utero, with a predisposition to generate tolerance through preferential Treg cell differentiation to self- and maternal antigens. Studies in neonates have underscored the importance of microbial pioneers on chronic inflammatory disease development in later life and is potentiated by bacterial metabolite effects on T cell immunity. I have generated preliminary data to support the existence of a microbiome in human fetal intestines and that fetal meconium communities dominated by Lactobacillus are enriched for regulatory T (Treg) cells in the adjacent intestinal lamina propria. Treg cells represent a key subset of adaptive immunity that promotes immunological tolerance. Thus, I hypothesize that the simple microbial communities found in the human fetal intestine are metabolically active and influence pre-natal immune tolerance development via their impact on Treg cell populations. To test this, I will determine the metabolic composition of fetal meconium and identify metabolites that are enriched in Lactobacillus-dominated samples. Enriched metabolites will be validated for induction of Treg cell differentiation ex vivo and genetic pathways for biosynthesis of these metabolites will be interrogated via metagenomic sequencing. Next, I will introduce commensal Lactobacillus johnsonii (LJ) that has been engineered to stably and constitutively express a GFP transgene (LJ-GFP) to pregnant dams vaginally or orally and localize LJ-GFP in murine fetal tissues. This will allow me to determine which route of commensal inoculation results in fetal intestinal colonization. I will then determine fetal intestinal metabolic alterations due to maternal supplementation just before delivery and assess the impact of LJ supplementation during pregnancy on post-natal induction of immune tolerance, using a cockroach allergen sensitization model post-weaning25. The completion of this study will serve as a fundamental cornerstone for developing microbial interventions during pregnancy to prevent a host of inflammatory diseases, such as childhood allergic sensitization and adult asthma.